RESUMEN
ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.
Asunto(s)
Antivirales/farmacología , Benzazepinas/farmacología , Citidina Trifosfato/análogos & derivados , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Farmacorresistencia Viral/genética , Niacinamida/análogos & derivados , Mutación Puntual , Proteínas Virales/antagonistas & inhibidores , Línea Celular Tumoral , Citidina Trifosfato/farmacología , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Combinación de Medicamentos , Sinergismo Farmacológico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Expresión Génica , Humanos , Niacinamida/farmacología , ARN Viral/antagonistas & inhibidores , ARN Viral/biosíntesis , ARN Viral/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Type 1 interferons (IFNs) have been shown to be efficacious against hepatitis C virus (HCV), hepatitis B virus (HBV), and some cancers with a significant drawback of short drug exposure. We have significantly improved the pharmacokinetic (PK) of consensus interferon (CIFN) by glycoengineering. We generated AL-624 by introducing 4 glycosylation sites. AL-624 was expressed, purified, and fractionated to yield 2-Gly, 3-Gly, and 4-Gly. In a rat PK study, AL-624 4-Gly exhibited a 6-fold increase of area under curve (AUC) and more than an 11-fold increase in time to half life (T1/2) over CIFN, suggesting the potential for weekly dosing (QW). In Yellow fever virus hamster model, QW of 4-Gly achieved similar efficacy to daily dosing (QD) CIFN and QW Peg-IFN-α-2a in overall survival rate and reduction of alanine aminotransferase (ALT) level. Further refinement resulted in development of AL-683 by addition of external glycosylation sites and its mouse homologue. AL-683 maintains undiminished biological potency in HCV replicon. In mouse PK/pharmacodynamic (PD) studies, AL-683 homologue has a â¼37-fold improvement in T1/2 and a â¼33-fold improvement in AUC when compared with the unglycosylated mouse IFN-α-1. Significantly improved PD responses were also observed. The significant improvement of AL-683 PK over AL-624 suggests a bimonthly dosing regimen for AL-683. The possibility for once-a-month dosing could be realized by further optimization of manufacturing conditions.
